Mechanistic insights into Variants of Uncertain Significance (VUS) using novel EGFR variants as a paradigm Investigators: Christine M. Lovly, MD, PhD and Jens Meiler, PhD The prospective identification and rational therapeutic targeting of tumor genomic alterations has revolutionized the care of patients with lung cancer and is now the accepted standard of care for patients with this disease and with other tumor types. With the advent of sophisticated tumor genotyping, the discovery of novel genetic variants is accelerating. In order to realize the promise of precision medicine, there is an urgent need to define the actionability of these variants to select targeted inhibitors. The objective of this proposal is to develop a novel, data-driven paradigm for characterizing genomic Variants of Uncertain Significance (VUS) and generating actionable hypotheses about their functions. For this purpose, we propose an innovative `Personalized Structural Biology' approach. The central hypothesis of this paradigm is that VUS can be best understood by placing the mutation into the context of protein structures and inferring from the structural consequences of the mutation on function, phenotype, and drug sensitivity. By analyzing the tumors of patients with lung cancer, we have identified three EGFR genomic alterations that have not previously been reported: 1) EGFR exon 18-25 Kinase Domain Duplication, 2) EGFR- RAD51 fusions, and 3) EGFR transmembrane domain mutations. Importantly, each of these EGFR variants was reported as a VUS on clinical genotyping reports because there were no data regarding the sensitivity of the mutated proteins to EGFR inhibitors now used in clinical practice. We sought to study these EGFR VUS in an effort to understand on a fundamental mechanistic level how they activate the EGF receptor to promote oncogenesis. We will integrate structural and computational modeling with various biochemical, molecular, cell based, and in vivo approaches to investigate the functional effects of these three distinct alterations. Through these studies, we expect to define previously unrecognized mechanisms of oncogenesis in lung cancer defined by these novel and recurrently detected EGFR variants. Importantly, understanding the structural and functional consequences of these EGFR variants is expected to provide novel insights into ErbB receptor biology and reveal new uses for FDA approved agents in lung cancer and many other tumor types which harbor ErbB (EGFR/HER2/HER3/HER4) alterations. Furthermore, a key deliverable of the proposed studies is the development of an innovative, integrated in silico pipeline that is applicable to VUS in any type of cancer, which we will make freely available via RosettaCommons (www.rosettacommons.org). Thereby, the impact of the proposed research will go beyond that of the three EGFR variants discussed in this proposal. With the recent FDA approval of NGS-based tumor testing, the problem of VUS will continue to grow as these large (>300) gene panel clinical diagnostic assays are reaching higher volumes of patients. Therefore, it is imperative the field generate and systematically integrate such `personalized structural biology' methods to understand the functional significance of VUS in order to best serve all cancer patients.